Eukaryotes have evolved numerous innate immune defenses against invading pathogens. One such defense is constituted by the APOBEC3 family of cytidine deaminases, a family of nine genes in the human, at least two of which (APOBEC3F and APOBEC3G) play a key role in defense against HIV-1. One family member, APOBEC3A (hA3A) had been an orphan that lacked activity against lentiviruses. We discovered that hA3A can potently inhibit two very different genetic elements: (i) a human parvovirus adeno-associated virus (AAV), and (ii) human and mouse endogenous retroelements. Paradoxically, although hA3A is an active cytidine deaminase, mutations have not been detected in retrotransposon or AAV DNA. A combination of biochemistry, genetics and cellular assays will be used to characterize hA3A and determine its mode of inhibition for parvoviruses and retroelements. We have purified recombinant hA3A and developed assays that will enable biochemical characterization of its enzymatic activity, DNA binding and protein complexes. Mutants that lack deaminase activity will be combined with cell-based and in vitro assays to study the mechanism of inhibition of AAV replication and retrotransposition. The studies proposed in this application will shed light on new modes of innate antiviral defense and will specifically determine the mechanism by which hA3A mediates its antiviral effect. The fact that hA3A has a single active site deaminase domain makes it particularly tractable for structure-function analysis. Parvoviruses provide a new context in which to examine the effects of APOBEC proteins and the mechanism of the deamination-independent antiviral activity. This combination provides a unique niche in which to examine APOBEC functions and unravel mechanisms responsible for a range of antiviral activities. PUBLIC HEALTH RELEVANCE: Eukaryotes have evolved numerous innate immune defenses against invading pathogens. The APOBEC family consists of related enzymes that have the ability to alter the DNA of viral pathogens and inhibit their growth. This project will investigate one member of the APOBEC3 family that is active against two very different sorts of pathogens: the parvoviruses and endogenous viruses called retrotransposons.